The molecular control and evolution of bacterial shapecnls.lanl.gov/annual27/Speaker Talks/Weibel CNLS May 2007.pdfThe molecular control and evolution of bacterial shape [email protected]

Doug Weibel

The molecular control and evolution of bacterial shape

[email protected]

Dept. of BiochemistryUniversity of Wisconsin-Madison

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mailto:[email protected]

mailto:[email protected]

Overview

Introduction to bacteriaSpatial organization of intracellular components

Conclusion and outlook

Part iMolecular control of cell shape in bacteria

Part ii (brief)Evolution of bacterial cell shape

Part iii (brief)Shape vs hydrodynamics at low Re

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Overview

Introduction to bacteriaSpatial organization of intracellular components

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Spatial organization: three fundamentals

10 μm

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Spatial organization: three fundamentalsi. What is the organization of machinery in cells?

10 μm

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Spatial organization: three fundamentalsi. What is the organization of machinery in cells?ii. How is organization established (mol. level)?

10 μm

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Spatial organization: three fundamentalsi. What is the organization of machinery in cells?ii. How is organization established (mol. level)?iii. How is organization replicated?

10 μm

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Torsten Wittman, UCSF Nikon

10 μm

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Our understanding in eukaryotic cells is emerging

Torsten Wittman, UCSF Nikon

10 μm

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Spatial organization in bacteria

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Our understanding is just beginning to emerge


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Bacteria are small; tools are still not available

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10 μm

Swiss 3T3 fibroblast

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10 μm


Cell of E. coli

~1 μm

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10 μm


Cell of E. coli

~1 μm

d = 0.61 λ/N.A.

NAmax (100x)=1.49

λmin=365 nm

dmax>150 nm

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Intracellular organization in bacteria

YFP-MreB chimera expressed in E. coli (Weibel)

Cell border

500 nm

GFP-FtsZ chimera expressed in E. coli (Beckwith)

GFP-Crescentin chimera expressed in C. crescentus (Jacobs-Wagner)

ParM chimera in vitro (Mullinsand Weibel)

5 μm 1 μm

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Cell border

500 nm




Actin homologs

5 μm 1 μm

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Cell border

500 nm




Actin homologs Tubulin homolog

5 μm 1 μm

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Cell border

500 nm




Actin homologs Tubulin homolog Intermediatefilament homolog

5 μm 1 μm

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Cell border

500 nm



Bacteria ‘share’ the major classes of cytoskeletalproteins found in eukaryotes


Actin homologs Tubulin homolog Intermediatefilament homolog

5 μm 1 μm

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Role of the bacterial cytoskeleton

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OrganizationPlays a role in cell shape

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DynamicsFunction?

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DynamicsFunction?

EvolutionUnknown?

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DynamicsFunction?

EvolutionUnknown?

We want to understand:The molecular basis for cell shapeThe evolution of bacterial cell shape

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Bacterial cell shape: an esoteric area?

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Fundamental biological question

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Improves our systems level understanding of bacteria/microbes (recall: microbes account for ~50% of biomass on planet)

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Improves our systems level understanding of bacteria/microbes (recall: microbes account for ~50% of biomass on planet)

Applications to pathogenesis and infectious diseases

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Overview

Part iMolecular control of cell shape in bacteria

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cocci; bacilli; spirillium

Bacterial cell shape: some examples

crescents

flat, square plates

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Copyright 2004 Pearson Education

Bacterial cell shape: structure of the cell wall

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D-Ala

L-Ala

D-Glu

meso-DAP

OO

OH

HOO

O

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C

HO2C NH2

NAG NAM


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OO

OH

HOO

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C NH2

O

HO

OO

OH

HOO

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C NH2

O

HO

D-Ala

L-Ala

D-Glu

meso-DAP

OO

OH

HOO

O

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C

HO2C NH2

NAG NAM


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OO

OH

HOO

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C NH2

O

HO

OO

OH

HOO

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C NH2

O

HO

D-Ala

L-Ala

D-Glu

meso-DAP

OO

OH

HOO

O

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C

HO2C NH2

NAG NAM

transglycosylases transglycosylases


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transpeptidasestranspeptidases

OO

OH

HOO

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C NH2

O

HO

OO

OH

HOO

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C NH2

O

HO

D-Ala

L-Ala

D-Glu

meso-DAP

OO

OH

HOO

O

OH

HNO

H3C OHN

O CH3

O

ONH

H3C

H3C

HNO

CO2H

NHO

NH

CH3

O

HO2C

HO2C NH2

NAG NAM

transglycosylases transglycosylases


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Structure of peptidoglycan

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Mobashery, ND

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Peptidoglycan (PG): Cross-linked polysaccharideSingle molecule (~100,000 kD)~20 nm thick (Gram-negative)Mechanical properties of cell


Mobashery, ND

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Mechanical properties of the PG

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Mechanical properties of the PGIsolate PG from cells (Triton X-100)Measure properties of intact PG with a scanning probe

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T. Beveridge (1999) J. Bacteriology, 181, 6865

Properties of the PG: Perfectly elastic (no hysteresis)γpeptidoglycan: 2.5x107 N/m2

(γlatex: ~2.5x107 N/m2)

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Cell shape is controlled at the level of the PG

2. PG is synthesized in a specific orientation (sculpted into a specific shape during synthesis)

1. PG is reinforced with mechanical ‘struts’ (molded and held in place)

Two plausible mechanisms:

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Penicillin binding protein (PBP)

Does the orientation of the PG control shape?

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12 known PBPs in E. coli



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PBPs play a key role in:Elongation of cell wall (PBP2)Division/septation (PBP3)Cell shape?

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PBPs play a key role in:Elongation of cell wall (PBP2)Division/septation (PBP3)Cell shape?

PG with κlow

PG with κhigh

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Are PBPs moved/localized on a filament?

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PBP2 (synthesis of cylindrical walls) interacts with MreB


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MreB: a homolog of actin that forms helical (?) filament(s) in rod-shaped cells

A GFP-MreB chimeraexpressed in B. subtilis(J. Errington et al.)

A mesoscale model; an elasticspring inserted in a flexible plastic tube (Weibel)



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MreB: a homolog of actin that forms helical (?) filament(s) in rod-shaped cells

A GFP-MreB chimeraexpressed in B. subtilis(J. Errington et al.)

A mesoscale model; an elasticspring inserted in a flexible plastic tube (Weibel)


MreB may play a key role in controlling:Spatial organization within the cellCell shape


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Method for dissecting role of proteins in cell shape

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We are systematically studying proteins (and protein interactions) that play a role in controlling cell shape


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We want to understand how perturbations in protein levels and organization influence cell shape

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To make this approach possible we have developed a technique for manipulating cell shape

We want to understand how perturbations in protein levels and organization influence cell shape

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hydrogel microchamber

cell growth release

Materials-based approach: (for example, rod-to-crescent)

S

N

CO2HH3C

NHH

O

NH2

O

Controlling cell shape

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hydrogel microchamber

cell growth release

Materials-based approach: (for example, rod-to-crescent)

S

N

CO2HH3C

NHH

O

NH2

O

Controlling cell shape

Steps:1. ‘Customize’ microchambers2. Seed cells3. Grow into filaments (antibiotic or Parab-FtsZ)4. Release by removing ‘ceiling’

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Filamentous cells of E. coli (filaments)

a cell of E. coli

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a cell of E. coli

(cephalexin)S

N

CO2HH3C

NHH

O

NH2

O

PBP3

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a cell of E. coli

a filament of E. coli

(cephalexin)S

N

CO2HH3C

NHH

O

NH2

O

PBP3

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de Pedro et al. (1997) J. Bacteriol., 179, 2823

Distribution of peptidoglycan from original cell (dark spots)

a cell of E. coli

a filament of E. coli

(cephalexin)S

N

CO2HH3C

NHH

O

NH2

O

PBP3

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transparency photomask1. expose (UV)

2. develop

3. cast hot agarose

4. cool

Fabricating molds for engineering cell shape

photoreactive polymeron Si wafer

micropatternedagarose

polymer patterned on Si wafer

coverslip

agarosecells

1. flip over

2. add cells

add transparentpolymer ‘ceiling’

polymer ceiling press ceilinginto contact

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Cells of E. coli in hydrogel containers are motile and metabolically active

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21


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Hydrogel walls are ‘transparent’ to nutrients, ions, gas, metabolic waste

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8 μm

(di

amet

er o

f cha

mbe

r)

3 μm (width of channel)

growth over 1.5 hr; 37 ℃

Growth of cells in microchambers

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8 μm

(di

amet

er o

f cha

mbe

r)




doubling timemold: 41 ± 4 min

doubling timesoln: ~40 min

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8 μm

(di

amet

er o

f cha

mbe

r)



Cells retain all of the phenotypes we observe in liquid cultures


doubling timemold: 41 ± 4 min

doubling timesoln: ~40 min

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zigzags

Geometry of walls controls cell shape

sinusoids

circles

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zigzags


sinusoids

circles γmold >> γcell

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zigzags


sinusoids

circles

Cells appear to grow in an orientation that minimizesstress placed on the cell

γmold >> γcell

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Cells retain their shape after release

circles

Grow cells of E. coli (rods) in chambers

spirals/helices

Cells in liquid (with cephalexin)

Release

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Cells retain their shape after release

circles

Grow cells of E. coli (rods) in chambers

spirals/helices

Cells in liquid (with cephalexin)

Release

Cell shape has been manipulated by the application of mechanical constraints

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Dimensions of circular chambers: 8 μm diameter; 2.2 μm tall

Hysteresis accompanies release of cells

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After release

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Hysteresis indicates strain on the cell wall


After release

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Hysteresis and calculation of γcell

Serial: measurement of γcell

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Hysteresis and calculation of γcell

Serial: measurement of γcell

Calculate stress/strain curve using the ‘relaxation’ of shape after cell release

Parallel measurement of γcell

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t=0 after release t=30 min

20 μm

t=0 after release t=45 min

10 μm

Engineered cells retain their shape during growth in liquid

d=14 μm d=20 μm

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Washing out cephalexin causes cells to septate

Cells septate in the absence of cephalexin

Motile cells retain their shape

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Overview

Part ii (brief)Evolution of bacterial cell shape

Part iii (brief)Shape vs hydrodynamics at low Re

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Why do cells have defined shapes?

Evolution of bacterial shape

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What is the evolutionary advantage of one shape over

another? cocci vs. bacilli vs. spirillium and so on...

30





Do we understand what bacteria really look like in their

native habitats?

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Do we understand what bacteria really look like in their

native habitats?

Bacterial motility is one interesting case to consider

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E. coli motility requires the bundling of flagella

RunTumble

Protonic Nanomachine Projecthttp://www.npn.jst.go.jp/index.html

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Bacterial motility and shape

E. coli (wild type) vtrans~10-20 μm sec-1

Turner & Berg

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Crescents vtrans~3-4 μm sec-1


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Crescents vtrans~3-4 μm sec-1


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Spirals vtrans~0 μm sec-1


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Spirals vtrans~0 μm sec-1


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Extended spirals vtrans ~5 μm sec-1


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Extended spirals vtrans ~5 μm sec-1


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Hydrodynamics on motilityAre we observing hydrodynamic effects on the cell body?

36


Slender body theorum

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translation

a

b

f = 4πηaln 2a 1

b 2_

a

b

translation

f = 8πηaln 2a 1

b 2+

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Probably not


translation

a

b

f = 4πηaln 2a 1

b 2_

a

b

translation

f = 8πηaln 2a 1

b 2+

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Hydrodynamics on motility

Bundling

Are we observing hydrodynamic effects on the flagellum?

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Bundling


Probably

37


Bundling


Probably

Why have multiple flagella if curvature prevents themfrom bundling?

Flagella are ‘expensive’ from an energetic standpoint

37


Bundling


Probably

Why have multiple flagella if curvature prevents themfrom bundling?

Flagella are ‘expensive’ from an energetic standpoint

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Have motile cells evolved shapes that maximize the bundling of flagella?

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Vibrio have evolved into crescents. 21/24 species of Vibrio we have looked at have a crescent shape and a single, polar flagellum

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Vibrio have evolved into crescents. 21/24 species of Vibrio we have looked at have a crescent shape and a single, polar flagellum

E. coli and other multi-flagellated strains of motile bacteria are rod-shaped

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Conclusion

Relationship between intracellular organization and bacterial cell shape

Our approach is to develop new capabilities for manipulating and studying bacterial cells

New techniques for intracellular imaging

Theoretical models for studying the evolution of shape

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NIHFunding

JSPS

Matt CopelandHannah TusonCorinne LipscombBasu BhattacharyaJoe MolendaCharlie BurnsSean McMaster

George Whitesides (Harvard)

Collaborators

Shoji Takeuchi (Tokyo)

Acknowledgements

Group

Willow DiLuzio (Harvard)

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Documents

The molecular control and evolution of bacterial shapecnls.lanl.gov/annual27/Speaker Talks/Weibel CNLS May 2007.pdfThe molecular control and evolution of bacterial shape [email protected]